Developing a novel immune infiltration-associated mitophagy prediction model for amyotrophic lateral sclerosis using bioinformatics strategies.

Background: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, which leads to muscle weakness and eventual paralysis. Numerous studies have indicated that mitophagy and immune inflammation have a significant impact on the onset and advancement of ALS. Nevertheless, the possible diagnostic and prognostic significance of mitophagy-related genes associated with immune infiltration in ALS is uncertain. The purpose of this study is to create a predictive model for ALS using genes linked with mitophagy-associated immune infiltration. Methods: ALS gene expression profiles were downloaded from the Gene Expression Omnibus (GEO) database. Univariate Cox analysis and machine learning methods were applied to analyze mitophagy-associated genes and develop a prognostic risk score model. Subsequently, functional and immune infiltration analyses were conducted to study the biological attributes and immune cell enrichment in individuals with ALS. Additionally, validation of identified feature genes in the prediction model was performed using ALS mouse models and ALS patients. Results: In this study, a comprehensive analysis revealed the identification of 22 mitophagy-related differential expression genes and 40 prognostic genes. Additionally, an 18-gene prognostic signature was identified with machine learning, which was utilized to construct a prognostic risk score model. Functional enrichment analysis demonstrated the enrichment of various pathways, including oxidative phosphorylation, unfolded proteins, KRAS, and mTOR signaling pathways, as well as other immune-related pathways. The analysis of immune infiltration revealed notable distinctions in certain congenital immune cells and adaptive immune cells between the low-risk and high-risk groups, particularly concerning the T lymphocyte subgroup. ALS mouse models and ALS clinical samples demonstrated consistent expression levels of four mitophagy-related immune infiltration genes (BCKDHA, JTB, KYNU, and GTF2H5) with the results of bioinformatics analysis. Conclusion: This study has successfully devised and verified a pioneering prognostic predictive risk score for ALS, utilizing eighteen mitophagy-related genes. Furthermore, the findings indicate that four of these genes exhibit promising roles in the context of ALS prognostic.

Use of Muscle Ultrasonography in Morphofunctional Assessment of Amyotrophic Lateral Sclerosis (ALS).

Amyotrophic lateral sclerosis (ALS) is a progressive disease with a high prevalence of malnutrition that can influence prognosis. The main objective of this study is to compare the validity of muscle ultrasonography in the diagnosis of malnutrition and the prognosis of patients with ALS. METHODS: This is a prospective observational study that analyzes the nutritional status of patients at the beginning of nutritional monitoring. The morphofunctional assessment included the examination of anthropometric variables such as weight, height, body mass index (BMI), arm circumference, and calf circumference. Additionally, electrical bioimpedanciometry (BIA) was used to measure electrical parameters and estimate other relevant metrics. Muscle ultrasonography® (quadriceps rectus femoris (QRF)) assessed muscle mass parameters, including muscle area index (MARAI), anteroposterior diameter of the QRF (Y-axis) (cm), transverse diameter of the QRF (X-axis) (cm), and the sum of the quadriceps thickness (RF+VI) (cm), as well as muscle quality parameters such as echogenicity and the Y-X index. RESULTS: A total of 37 patients diagnosed with amyotrophic lateral sclerosis (ALS) were included in this study. Of these patients, 51.4% were men. The mean age was 64.27 (12.59) years. A total of 54.1% of the patients had a bulbar onset of amyotrophic lateral sclerosis, and 45.9% had spinal onset. The percentage of subjects with malnutrition diagnosed by the Global Leadership Initiative on Malnutrition (GLIM) criteria was 45.9% of patients. There was a direct correlation between muscle mass parameters assessed by muscle ultrasonography (RF+VI) and active mass markers measured by bioimpedanciometry (body cellular mass index (BCMI) (r = 0.62; p < 0.01), fat-free mass index (FFMI) (r = 0.75; p < 0.01), and appendicular skeletal mass index (ASMI) (r = 0.69; p < 0.01)). There was a direct correlation between echogenicity and resistance (r = 0.44; p = 0.02), as well as between the fat-free mass index and the Y-X index (r = 0.36; p = 0.14). Additionally, there was a negative correlation between echogenicity and BCMI (r = -0.46; p < 0.01) and ASMI (r = 0.34; p = 0.06). Patients with low quadriceps thickness (male < 2.49 cm; female < 1.84 cm) showed an increased risk of hospital admission adjusted by age, sex, and presence of dysphagia (OR: 7.84 (CI 95%: 1.09-56.07); p-value = 0.04), and patients with low-quality mass (Y-X index < 0.35) had a higher risk of hospital admission adjusted by age, sex, and presence of dysphagia (OR: 19.83 (CI 95%: 1.77-222.46); p-value = 0.02). CONCLUSIONS: In patients with ALS, ultrasonography echogenicity was inversely related to BCMI, FFMI, and ASMI, and the Y-X index was directly related to FFMI. The lowest quartiles of quadriceps thickness and Y-X index are risk factors for hospital admission.

Optimizing breathlessness management in amyotrophic lateral sclerosis: insights from a comprehensive systematic review.

BACKGROUND: Breathlessness is a prevalent symptom affecting the quality of life (QOL) of Amyotrophic Lateral Sclerosis (ALS) patients. This systematic review explored the interventions for controlling breathlessness in ALS patients, emphasizing palliative care (PALC), non-invasive ventilation (NIV), opioids, and non-pharmacological strategies. METHODS: A comprehensive search of PubMed, Cochrane Library, and Web of Science databases was conducted. Eligibility criteria encompassed adults with ALS or motor neuron disease experiencing breathlessness. Outcomes included QOL and symptom control. Study designs comprised qualitative studies, cohort studies, and randomized controlled trials. RESULTS: Eight studies were included, most exhibiting low bias risk, comprising one randomized controlled trial, three cohort studies, two comparative retrospective studies, and two qualitative studies (interviews). Most studies originated from Europe, with one from the United States of America. The participants totaled 3423, with ALS patients constituting 95.6%. PALC consultations significantly improved symptom assessment, advance care planning, and discussions about goals of care. NIV demonstrated efficacy in managing breathlessness, with considerations for device limitations. Opioids were effective, though predominantly studied in non-ALS patients. Non-pharmacological strategies varied in efficacy among patients. CONCLUSION: The findings underscore the need for individualized approaches in managing breathlessness in ALS. PALC, NIV, opioids, and non-pharmacological strategies each play a role, with unique considerations. Further research, especially ALS-specific self-management studies, is warranted.

Peripheral Immune Profiles Predict ALS Progression in an Age- and Sex-Dependent Manner.

BACKGROUND AND OBJECTIVES: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease whose pathobiology associates with peripheral blood immune cell levels and activation patterns in an age and sex-dependent manner. This study's objective was to identify immune profile associations with ALS progression, whether the associations are age and sex-specific, and whether immune profiles can predict a future disease course. METHODS: Flow cytometry immune profiles (a combination of 22 peripheral blood immune markers) were generated for 241 participants with ALS and linked to ALS progression, using progression-free survival, which is a composite combining the revised ALS Functional Rating Scale and survival. Participants were first grouped by immune profiles using unsupervised hierarchical clustering, and clusters were associated with subsequent progression-free survival. Next, individual immune markers were associated with progression-free survival using least absolute shrinkage and selection operator-Cox regression. Analyses were stratified by age and sex to identify demographic-specific immune mechanisms. Finally, random forest determined the predictive power of immune profiles on ALS progression in the whole population and again stratified by age and sex. RESULTS: Progression-free survival differed between clusters of participants with similar immune profiles, particularly reduced natural killer (NK)-cell activation associated with slower progression. Individual markers such as neutrophil levels and NK-cell NKp46 expression associated with faster ALS progression while overall NK-cell levels and NK-cell subpopulations associated with slower progression; the strength of these associations varied by age and sex. Adding these immune markers to prediction models dramatically increased short-term prediction compared with routine clinical prognostic variables alone, and the addition of NK-cell markers further improved the prediction accuracy in female participants. DISCUSSION: Specific immune profiles likely contribute to ALS progression in an age and sex-dependent manner, and peripheral immune markers enhance the prediction of short-term clinical outcomes. These findings suggest a complex milieu of immune profiles associated with ALS progression, and more detailed immunophenotyping in ALS will facilitate personalized immunotherapeutics in ALS.

A Potential Role of Interleukin-5 in the Pathogenesis and Progression of Amyotrophic Lateral Sclerosis: A New Molecular Perspective.

Cumulative data suggest that neuroinflammation plays a prominent role in amyotrophic lateral sclerosis (ALS) pathogenesis. The purpose of this work was to assess if patients with ALS present a specific peripheral cytokine profile and if it correlates with neurological disability assessed by ALSFRS-R, the rate of disease progression, and the pattern of disease progression (horizontal spreading [HSP] versus vertical spreading [VSP]). We determined the levels of 15 cytokines in the blood of 59 patients with ALS and 40 controls. We identified a positive correlation between levels of pro-inflammatory cytokines (interleukin [IL]-17F, IL-33, IL-31) and the age of ALS patients, as well as a positive correlation between IL-12p/70 and survival from ALS onset and ALS diagnosis. Additionally, there was a positive correlation between the ALSFRS-R score in the upper limb and respiratory domain and IL-5 levels. In our ALS cohort, the spreading pattern was 42% horizontal and 58% vertical, with patients with VSP showing a faster rate of ALS progression. Furthermore, we identified a negative correlation between IL-5 levels and the rate of disease progression, as well as a positive correlation between IL-5 and HSP of ALS. To the best of our knowledge, this is the first study reporting a "protective" role of IL-5 in ALS.

Neuroinflammation in Neurodegenerative Disorders: Current Knowledge and Therapeutic Implications.

Neurodegenerative disorders (NDs) have become increasingly common during the past three decades. Approximately 15% of the total population of the world is affected by some form of NDs, resulting in physical and cognitive disability. The most common NDs include Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and Huntington's disease. Although NDs are caused by a complex interaction of genetic, environmental, and lifestyle variables, neuroinflammation is known to be associated with all NDs, often leading to permanent damage to neurons of the central nervous system. Furthermore, numerous emerging pieces of evidence have demonstrated that inflammation not only supports the progression of NDs but can also serve as an initiator. Hence, various medicines capable of preventing or reducing neuroinflammation have been investigated as ND treatments. While anti-inflammatory medicine has shown promising benefits in several preclinical models, clinical outcomes are often questionable. In this review, we discuss various NDs with their current treatment strategies, the role of neuroinflammation in the pathophysiology of NDs, and the use of anti-inflammatory agents as a potential therapeutic option.

C9ORF72 patient-derived endothelial cells drive blood-brain barrier disruption and contribute to neurotoxicity.

The blood-brain barrier (BBB) serves as a highly intricate and dynamic interface connecting the brain and the bloodstream, playing a vital role in maintaining brain homeostasis. BBB dysfunction has been associated with multiple neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS); however, the role of the BBB in neurodegeneration is understudied. We developed an ALS patient-derived model of the BBB by using cells derived from 5 patient donors carrying C9ORF72 mutations. Brain microvascular endothelial-like cells (BMEC-like cells) derived from C9ORF72-ALS patients showed altered gene expression, compromised barrier integrity, and increased P-glycoprotein transporter activity. In addition, mitochondrial metabolic tests demonstrated that C9ORF72-ALS BMECs display a significant decrease in basal glycolysis accompanied by increased basal and ATP-linked respiration. Moreover, our study reveals that C9-ALS derived astrocytes can further affect BMECs function and affect the expression of the glucose transporter Glut-1. Finally, C9ORF72 patient-derived BMECs form leaky barriers through a cell-autonomous mechanism and have neurotoxic properties towards motor neurons.

Analysis of Heart Rate Variability in Individuals Affected by Amyotrophic Lateral Sclerosis.

INTRODUCTION: Amyotrophic lateral sclerosis (ALS) produces alterations in the autonomic nervous system (ANS), which explains the cardiac manifestations observed in patients. The assessment of heart rate variability (HRV) is what best reflects the activity of the ANS on heart rate. The Polar H7 Bluetooth® device proves to be a non-invasive and much faster technology than existing alternatives for this purpose. OBJECTIVE: The goal of this study is to determine HRV using Polar H7 Bluetooth technology in ALS patients, comparing the obtained measurements with values from healthy individuals. METHOD: The sample consisted of 124 participants: 68 diagnosed with ALS and 56 healthy individuals. Using Polar H7 Bluetooth technology and the ELITE HRV application, various HRV measurements were determined for all participants, specifically the HRV index, RMSSD, RMSSD LN, SDNN index, PNN50, LF, HF, LF/HF ratio, HR average, and HF peak frequency. RESULTS: Statistically significant differences were observed between ALS patients and healthy individuals in the HRV index, RMSSD, RMSSD LN, SDNN index, PNN50, HF, and LF, where healthy individuals exhibited higher scores. For the HR average, the ALS group showed a higher value. Values were similar when comparing men and women with ALS, with only a higher HF peak frequency observed in women. CONCLUSION: The Polar H7 Bluetooth® device is effective in determining heart rate variability alterations in ALS, being a promising prognostic tool for the disease.

Reversal of C9orf72 mutation-induced transcriptional dysregulation and pathology in cultured human neurons by allele-specific excision.

Efforts to genetically reverse C9orf72 pathology have been hampered by our incomplete understanding of the regulation of this complex locus. We generated five different genomic excisions at the C9orf72 locus in a patient-derived induced pluripotent stem cell (iPSC) line and a non-diseased wild-type (WT) line (11 total isogenic lines), and examined gene expression and pathological hallmarks of C9 frontotemporal dementia/amyotrophic lateral sclerosis in motor neurons differentiated from these lines. Comparing the excisions in these isogenic series removed the confounding effects of different genomic backgrounds and allowed us to probe the effects of specific genomic changes. A coding single nucleotide polymorphism in the patient cell line allowed us to distinguish transcripts from the normal vs. mutant allele. Using digital droplet PCR (ddPCR), we determined that transcription from the mutant allele is upregulated at least 10-fold, and that sense transcription is independently regulated from each allele. Surprisingly, excision of the WT allele increased pathologic dipeptide repeat poly-GP expression from the mutant allele. Importantly, a single allele was sufficient to supply a normal amount of protein, suggesting that the C9orf72 gene is haplo-sufficient in induced motor neurons. Excision of the mutant repeat expansion reverted all pathology (RNA abnormalities, dipeptide repeat production, and TDP-43 pathology) and improved electrophysiological function, whereas silencing sense expression did not eliminate all dipeptide repeat proteins, presumably because of the antisense expression. These data increase our understanding of C9orf72 gene regulation and inform gene therapy approaches, including antisense oligonucleotides (ASOs) and CRISPR gene editing.

Animal Models of FUS-Proteinopathy: A Systematic Review.

Mutations that disrupt the function of the DNA/RNA-binding protein FUS could cause amyotrophic lateral sclerosis (ALS) and other neurodegenerative diseases. One of the key features in ALS pathogenesis is the formation of insoluble protein aggregates containing aberrant isoforms of the FUS protein in the cytoplasm of upper and lower motor neurons. Reproduction of human pathology in animal models is the main tool for studying FUS-associated pathology and searching for potential therapeutic agents for ALS treatment. In this review, we provide a systematic analysis of the role of FUS protein in ALS pathogenesis and an overview of the results of modelling FUS-proteinopathy in animals.

KCNJ2 inhibition mitigates mechanical injury in a human brain organoid model of traumatic brain injury.

Traumatic brain injury (TBI) strongly correlates with neurodegenerative disease. However, it remains unclear which neurodegenerative mechanisms are intrinsic to the brain and which strategies most potently mitigate these processes. We developed a high-intensity ultrasound platform to inflict mechanical injury to induced pluripotent stem cell (iPSC)-derived cortical organoids. Mechanically injured organoids elicit classic hallmarks of TBI, including neuronal death, tau phosphorylation, and TDP-43 nuclear egress. We found that deep-layer neurons were particularly vulnerable to injury and that TDP-43 proteinopathy promotes cell death. Injured organoids derived from C9ORF72 amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD) patients displayed exacerbated TDP-43 dysfunction. Using genome-wide CRISPR interference screening, we identified a mechanosensory channel, KCNJ2, whose inhibition potently mitigated neurodegenerative processes in vitro and in vivo, including in C9ORF72 ALS/FTD organoids. Thus, targeting KCNJ2 may reduce acute neuronal death after brain injury, and we present a scalable, genetically flexible cerebral organoid model that may enable the identification of additional modifiers of mechanical stress.

[Neuropathology of the Neurodegenerative Diseases].

A definite diagnosis of neurodegenerative diseases is required for neuropathological examination during an autopsy. Each neurodegenerative disease has specific vulnerable regions and affected systems (system degeneration), and is typified by an accumulation of abnormal protein with the formation of characteristic morphological aggregates in the nerve and glial cells, called proteinopathy. The most common neurodegenerative diseases are tauopathy, such as progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and Pick's disease (PiD); α-synucleinopathy, including multiple system atrophy (MSA); and TAR DNA-binding protein of 43 kDa (TDP-43) proteinopathy, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). PSP and CBD show characteristic tau-positive astrocytic inclusions known as tufted astrocytes and astrocytic plaques, respectively. PiD shows tau-positive neuronal inclusions termed Pick bodies. MSA is characterized by α-synuclein-positive oligodendroglial inclusions, called glial cytoplasmic inclusions. ALS- and FTLD-TDP show TDP-43-positive neuronal inclusions, such as skein-like and round inclusions. Huntington's disease shows polyglutamine-positive neuronal inclusions, and Creutzfeldt-Jakob disease shows diffuse deposition of granular prions in the neuropil. The atypical proteins in these diseases have abnormal conformational properties. A comprehensive comparison of the clinical findings and neuropathological observations, including neuroanatomy and images acquired during life, is important to improve the sensitivity of clinical diagnosis.

Phosphorylation of the compartmentalized PKA substrate TAF15 regulates RNA-protein interactions.

Spatiotemporal-controlled second messengers alter molecular interactions of central signaling nodes for ensuring physiological signal transmission. One prototypical second messenger molecule which modulates kinase signal transmission is the cyclic-adenosine monophosphate (cAMP). The main proteinogenic cellular effectors of cAMP are compartmentalized protein kinase A (PKA) complexes. Their cell-type specific compositions precisely coordinate substrate phosphorylation and proper signal propagation which is indispensable for numerous cell-type specific functions. Here we present evidence that TAF15, which is implicated in the etiology of amyotrophic lateral sclerosis, represents a novel nuclear PKA substrate. In cross-linking and immunoprecipitation experiments (iCLIP) we showed that TAF15 phosphorylation alters the binding to target transcripts related to mRNA maturation, splicing and protein-binding related functions. TAF15 appears to be one of multiple PKA substrates that undergo RNA-binding dynamics upon phosphorylation. We observed that the activation of the cAMP-PKA signaling axis caused a change in the composition of a collection of RNA species that interact with TAF15. This observation appears to be a broader principle in the regulation of molecular interactions, as we identified a significant enrichment of RNA-binding proteins within endogenous PKA complexes. We assume that phosphorylation of RNA-binding domains adds another layer of regulation to binary protein-RNAs interactions with consequences to RNA features including binding specificities, localization, abundance and composition.

Evaluation of cyanotoxin L-BMAA effect on α-synuclein and TDP43 proteinopathy.

The complex interplay between genetic and environmental factors is considered the cause of neurodegenerative diseases including Parkinson's disease (PD) and Amyotrophic Lateral Sclerosis (ALS). Among the environmental factors, toxins produced by cyanobacteria have received much attention due to the significant increase in cyanobacteria growth worldwide. In particular, L-BMAA toxin, produced by diverse taxa of cyanobacteria, dinoflagellates and diatoms, has been extensively correlated to neurodegeneration. The molecular mechanism of L-BMAA neurotoxicity is still cryptic and far from being understood. In this research article, we have investigated the molecular pathways altered by L-BMAA exposure in cell systems, highlighting a significant increase in specific stress pathways and an impairment in autophagic processes. Interestingly, these changes lead to the accumulation of both α-synuclein and TDP43, which are correlated with PD and ALS proteinopathy, respectively. Finally, we were able to demonstrate specific alterations of TDP43 WT or pathological mutants with respect to protein accumulation, aggregation and cytoplasmic translocation, some of the typical features of both sporadic and familial ALS.

Modelos experimentales murinos en la esclerosis lateral amiotrófica. Puesta al día / Murine experimental models of amyotrophic lateral sclerosis: An update

Introducción: La esclerosis lateral amiotrófica (ELA) es una enfermedad neurodegenerativa, progresiva y de etiología desconocida caracterizada por la degeneración de motoneuronas superiores e inferiores. Aproximadamente el 90% de los casos de ELA son esporádicos, mientras que el 10% restante se consideran familiares. Independientemente de si son familiares o esporádicas, los pacientes desarrollan una debilidad progresiva, atrofia muscular con espasticidad y contracturas. Por lo general, la esperanza de vida en los pacientes de ELA es de 2 a 5 años. Desarrollo: Los modelos in vivo han ayudado a explicar la etiología y la patogénesis, así como los mecanismos de la ELA. Sin embargo, estos mecanismos no están del todo esclarecidos aún, por lo que los modelos experimentales son fundamentales para continuar con el estudio de los mismos, así como para la búsqueda de posibles dianas terapéuticas. A pesar de que el 90% de los casos son esporádicos, la mayoría de los modelos utilizados hasta la actualidad para estudiar la patogénesis están basados en las mutaciones genéticas asociadas a la enfermedad familiar, lo que provoca que la patogénesis de la ELA esporádica no sea aún conocida. Por tanto, sería fundamental el estudio de la enfermedad en modelos basados en la enfermedad esporádica. Conclusión: En el presente artículo se han revisado los principales modelos experimentales, tanto genéticos como esporádicos, utilizados en el estudio de esta enfermedad, enfocándonos en los que se han desarrollado utilizando el roedor como plataforma experimental.(AU)

Introduction: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease whose aetiology is unknown. It is characterised by upper and lower motor neuron degeneration. Approximately 90% of cases of ALS are sporadic, whereas the other 10% are familial. Regardless of whether the case is familial or sporadic, patients will develop progressive weakness, muscle atrophy with spasticity, and muscle contractures. Life expectancy of these patients is generally 2–5 years after diagnosis. Development: In vivo models have helped to clarify the aetiology and pathogenesis of ALS, as well as the mechanisms of the disease. However, as these mechanisms are not yet fully understood, experimental models are essential to the continued study of the pathogenesis of ALS, as well as in the search for possible therapeutic targets. Although 90% of cases are sporadic, most of the models used to study ALS pathogenesis are based on genetic mutations associated with the familial form of the disease; the pathogenesis of sporadic ALS remains unknown. Therefore, it would be critical to establish models based on the sporadic form. Conclusion: This article reviews the main genetic and sporadic experimental models used in the study of this disease, focusing on those that have been developed using rodents.(AU)

Anterior insula is more vulnerable than posterior insula to TDP-43 pathology in common dementias and ALS.

Based on the anatomic proximity, connectivity, and functional similarities between the anterior insula and amygdala, we tested the hypothesis that the anterior insula is an important focus in the progression of TDP-43 pathology in LATE-NC. Blinded to clinical and neuropathologic data, phospho-TDP (pTDP) inclusion pathology was assessed in paired anterior and posterior insula samples in 105 autopsied patients with Alzheimer disease, Lewy body disease, LATE-NC and hippocampal sclerosis (HS), amyotrophic lateral sclerosis (ALS), and other conditions. Insular pTDP pathology was present in 34.3% of the study cohort, most commonly as neuronal inclusions and/or short neurites in lamina II, and less commonly as subpial processes resembling those described in the amygdala region. Among positive samples, pTDP pathology was limited to the anterior insula (41.7%), or occurred in both anterior and posterior insula (58.3%); inclusion density was greater in anterior insula across all diseases (p < .001). pTDP pathology occurred in 46.7% of ALS samples, typically without a widespread TDP-43 proteinopathy. In LATE-NC, it was seen in 30.4% of samples (mostly LATE-NC stages 2 and 3), often co-occurring with basal forebrain pathology and comorbid HS, suggesting this is an important step in the evolution of this pathology beyond the medial temporal lobe.

A dynamic regulatory switch for phase separation of FUS protein: Zinc ions and zinc finger domain.

Zinc is an important trace element in the human body, and its homeostasis is closely related to amyotrophic lateral sclerosis (ALS). Cytoplasmic FUS proteins from patients with ALS aggregate their important pathologic markers. Liquid-liquid phase separation (LLPS) of FUS can lead to its aggregation. However, whether and how zinc homeostasis affects the aggregation of disease-associated FUS proteins in the cytoplasm remains unclear. Here, we found that zinc ion enhances LLPS and promotes the aggregation in the cytoplasm for FUS protein. In the FUS, the cysteine of the zinc finger (ZnF), recognizes and binds to zinc ions, reducing droplet mobility and enhancing protein aggregation in the cytoplasm. The mutation of FUS cysteine disrupts the dynamic regulatory switch of zinc ions and ZnF, resulting in insensitivity to zinc ions. These results suggest that the dynamic regulation of LLPS by binding with zinc ions may be a widespread mechanism and provide a new understanding of neurological diseases such as ALS and other ZnF protein-related diseases.

An aberrant fused in sarcoma liquid droplet of amyotrophic lateral sclerosis pathological variant, R495X, accelerates liquid-solid phase transition.

Intracellular aggregation of fused in sarcoma (FUS) is associated with the pathogenesis of familial amyotrophic lateral sclerosis (ALS). Under stress, FUS forms liquid droplets via liquid-liquid phase separation (LLPS). Two types of wild-type FUS LLPS exist in equilibrium: low-pressure LLPS (LP-LLPS) and high-pressure LLPS (HP-LLPS); the former dominates below 2 kbar and the latter over 2 kbar. Although several disease-type FUS variants have been identified, the molecular mechanism underlying accelerated cytoplasmic granule formation in ALS patients remains poorly understood. Herein, we report the reversible formation of the two LLPS states and the irreversible liquid-solid transition, namely droplet aging, of the ALS patient-type FUS variant R495X using fluorescence microscopy and ultraviolet-visible absorption spectroscopy combined with perturbations in pressure and temperature. Liquid-to-solid phase transition was accelerated in the HP-LLPS of R495X than in the wild-type variant; arginine slowed the aging of droplets at atmospheric conditions by inhibiting the formation of HP-LLPS more selectively compared to that of LP-LLPS. Our findings provide new insight into the mechanism by which R495X readily forms cytoplasmic aggregates. Targeting the aberrantly formed liquid droplets (the HP-LLPS state) of proteins with minimal impact on physiological functions could be a novel therapeutic strategy for LLPS-mediated protein diseases.

Choroid plexus enlargement in amyotrophic lateral sclerosis patients and its correlation with clinical disability and blood-CSF barrier permeability.

BACKGROUND: Using in vivo neuroimaging techniques, growing evidence has demonstrated that the choroid plexus (CP) volume is enlarged in patients with several neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. However, although animal and postmortem findings suggest that CP abnormalities are likely important pathological mechanisms underlying amyotrophic lateral sclerosis (ALS), the third most common neurodegenerative disease, no available study has been conducted to thoroughly assess CP abnormalities and their clinical relevance in vivo in ALS patients to date. Thus, we aimed to determine whether in vivo CP enlargement may occur in ALS patients. We also aimed to identify the relationships of CP volume with clinical disabilities and blood-CSF barrier (BCSFB) permeability in ALS patients. METHODS: In this retrospective study, based on structural MRI data, CP volume was assessed using a Gaussian mixture model and underwent further manual correction in 155 ALS patients and 105 age- and sex-matched HCs from October 2021 to April 2023. The ALS Functional Rating Scale-Revised (ALSFRS-R) was used to assess clinical disability. The CSF/serum albumin quotient (Qalb) was used to assess BCSFB permeability. Moreover, all the ALS patients completed genetic testing, and according to genetic testing, the ALS patients were further divided into genetic ALS subgroup and sporadic ALS subgroup. RESULTS: We found that compared with HCs, ALS patients had a significantly higher CP volume (p < 0.001). Moreover, compared with HCs, CP volume was significantly increased in both ALS patients with and without known genetic mutations after family-wise error correction (p = 0.006 and p < 0.001, respectively), while there were no significant differences between the two ALS groups. Furthermore, the CP volume was significantly correlated with the ALSFRS-r score (r = -0.226; p = 0.005) and the Qalb (r = 0.479; p < 0.001) in ALS patients. CONCLUSION: Our study first demonstrates CP enlargement in vivo in ALS patients, and continues to suggest an important pathogenetic role for CP abnormalities in ALS. Moreover, assessing CP volume is likely a noninvasive and easy-to-implement approach for screening BCSFB dysfunction in ALS patients.

Abundant transcriptomic alterations in the human cerebellum of patients with a C9orf72 repeat expansion.

The most prominent genetic cause of both amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) is a repeat expansion in the gene C9orf72. Importantly, the transcriptomic consequences of the C9orf72 repeat expansion remain largely unclear. Here, we used short-read RNA sequencing (RNAseq) to profile the cerebellar transcriptome, detecting alterations in patients with a C9orf72 repeat expansion. We focused on the cerebellum, since key C9orf72-related pathologies are abundant in this neuroanatomical region, yet TDP-43 pathology and neuronal loss are minimal. Consistent with previous work, we showed a reduction in the expression of the C9orf72 gene and an elevation in homeobox genes, when comparing patients with the expansion to both patients without the C9orf72 repeat expansion and control subjects. Interestingly, we identified more than 1000 alternative splicing events, including 4 in genes previously associated with ALS and/or FTLD. We also found an increase of cryptic splicing in C9orf72 patients compared to patients without the expansion and controls. Furthermore, we demonstrated that the expression level of select RNA-binding proteins is associated with cryptic splice junction inclusion. Overall, this study explores the presence of widespread transcriptomic changes in the cerebellum, a region not confounded by severe neurodegeneration, in post-mortem tissue from C9orf72 patients.